Manipulating DNA
Name _____________________________________
Introduction
Biotechnology is the manipulation of the biological capacity of cells and their components.
For thousands of years people have used biotechnology by using yeast to make flour into
bread and grapes into wine. Today, we are using biotechnology to study the basic processes
of life and diagnose illnesses and develop new treatments for disease.
Some of the tools of biotechnology are natural components of cells. Restriction
enzymes are made by bacteria to protect themselves from viruses. They inactivate the viral
DNA by cutting it in specific places. DNA ligase is an enzyme that exist in all cells and is
responsible for joining together strands of DNA. Scientists use restriction enzymes to cut
DNA at a specific sequence called recognition sites. They can rejoin the cut strands with DNA
ligase to make a new combination of genes. Recombinant DNA sequences contain genes
from two or more organisms.
Using the technique, researches have gained the ability to diagnose diseases such as
sickle cell anemia, cystic fibrosis, and Huntington’s disease early in the course of the disease.
Many researchers are also applying the techniques of biotechnology to find new treatments
for genetic diseases.
In this activity, you will use paper models to stimulate the cutting of DNA; you will also
model gel electrophoresis to analyze the DNA fragments produced. You will learn how these
techniques are carried out and also some of their applications.
As the illustration shows, the sequences of DNA can be written in many ways. However, the
simplest way is to use single-letter codes for the nucleotide bases: A(adenine), T(thymine),
C(cytosine), and G(guanine). The DNA sequence in the illustration can be written in its
shorthand fashion as:
A T T G C T A A C A
T A A C G A T T G T
1. What are the complementary bases for the DNA sequence below?
T A A G C C G T A G G T T G G A A C T C C
2. Write your own double stranded molecule, 20 base pairs long/
Procedure
Part I: Restriction Enzymes
A.There are now about 200 known restriction enzymes that cut DNA at specific recognition
sites. For example, the restriction enzyme Hind II recognizes the base sequence GTCGAC.
3. Complete the sequence below with the complementary bases and put parenthesis
around the Hind II recognition site.
T A A G C C G T C G A C T C G A A C T C C
Read the complementary strand in reverse and it indicates the Hind II recognition site.
B. When the restriction enzyme Hind II recognizes the sequence GTCGAC, it will cut the
DNA between the cytosine (C) and guanine (G) on both strands. Therefore, it will leave blunt
ends on the fragment:
Hind II
-GTCGAC- C A G C T G-

-G T C
GAC-C A G
CTG Blunt ends
The restriction enzyme Eco RI cuts its recognition site at nonadjacent points on the DNA
molecule, leaving “sticky” ends. Eco RI recognizes the base sequence GAATTC and cuts
this sequence between the guanine (G) and adenine (A) bases:
Eco RI
- GAATTC -CTTAAG -

-G
AATTC - CTTAA
GSticky ends
Therefore, a molecule of DNA cut at an Eco RI site would appear:
- CGTTATG - GCAATACTTAA
- AATTCGTAG –
- GCATC –
Sticky ends can bind to similar sticky ends from other Eco RI-digested DNA fragments. After
recombining, the ends are joined by DNA ligase, to form a new pattern of bases. By cutting
the DNA from 2 different organisms with the same enzyme and recombining with DNA ligase,
scientists make recombinant DNA.
4. Copy the sequence given below and complete the strand of complementary to it. On
both strands indicate the Eco RI restriction sites by putting parenthesis around them.
Remember to read the complementary strand in reverse.
G C C T C T A A G A A T T C A G T T C G
5. Once the Eco RI has cut the above DNA chain, how many fragments of DNA would
there be?
6. Would the ends be blunt or sticky?
7. How many bases would there be in each fragment? Note: When counting the length
of a DNA fragment, count only the number of bases on the upper strand.
C. Below you will see two sequences of DNA - DNA IA and DNA IB. ( DNA IB is a mutant
variation of DNA IA).
8. What is the difference between the two sequences?
9. On the sequence below identify the Eco RI recognition site on both sequences and
mark the sites where Eco RI would cut. You will use these sequences again in part II.
DNA IA
TTGCAGTCAGAAGAAT TCAACCTAGGAATTCTAAGCGC
T T C G T C A G T C T T C T T A A G T T G G A T C C T T A A GA T T C G C G
DNA IB
TTGCAGTCAGAAGAA GTCAACCTAGGAATTCTAAGCGC
T T C G T C A G T C T T C T T C A G T T G G A T C C T T A A GA T T C G C G
10. How many fragments of DNA were made from each sequence after digestion with Eco
RI?
11. What are the lengths (in base pairs) of the fragments from the DNA IA and DNA Ib
digestions?
Part II: Gel Electrophoresis
D. Scientists identify differences in DNA by measuring the length and number of fragments
created by digestion with restriction enzymes. A technique called gel electrophoresis is used
to separate fragments according to length. DNA fragments (cut with an appropriate restriction
enzyme) are placed on one end of a specially-prepared block of agarose which causes the
strands to migrate through the gel. (Since DNA molecules are negatively charged, they
migrate towards the positive electrode.) The agarose is like a sponge with small holes in it.
Therefore, the smaller DNA fragments can move through the gel at a faster rate than larger
fragments and the larger fragments are found nearer the point of origin. Scientist then use a
special stain to make the DNA fragments visible as bands. By counting the number of bands
researchers can tell how many fragments exist. By observing the distance each fragment has
migrated, they can determine how big each fragment is.
12. How many fragments are produced by cutting DNA X? By cutting DNA Y?
13 The fragments generated from DNA X and DNA Y in figure A were then analyzed
by gel electrophoresis. From the data in Figures B and C, calculate the sizes of the
fragments.
14. On the simulated gel, draw bands corresponding to the positions to which the Eco
RI digest DNA IA and DNA IB would migrate. The fragments of each type of DNA
should be in separate lanes and at distances from the origin proportional to the
number of bases in each fragment. (Remember that smaller fragments move further.)
From the following distances, determine how far your fragments will migrate: 40 base
pairs will migrate 2.5 cm; 30 bp will migrate 5 cm; 20 bp will migrate 7.5 cm; and 10 bp
will migrate 10 cm. Make sure all DNA is accounted for.